JP6764906B2 - Use of leptin to treat human adipose tissue atrophy and predisposition methods for the treatment - Google Patents

Abstract

Leptin, leptin analogs, and leptin derivatives are used to treat patients with lipodystrophy. Leptin is effective against lipodystrophy conditions for both genetic and acquired forms of the disease. A therapeutically effective amount of leptin can be administered in a variety of ways, including in a vector comprising nucleic acid sequences encoding leptin. Methods of the present invention contemplate administration of leptin, leptin analogs, and leptin derivatives to patients having approximately 4ng/ml or less before treatment.

Description

(Government interests)
The invention was partially supported by financial resources from NIH. The government has some rights in the present invention.
(Cross-reference of related applications)
The utility patent of this application claims priority over US provisional application No. 60/336394 (Depaoli et al.) Filed October 22, 2001, the disclosure of which is incorporated herein by reference.
(Technical field to which the invention belongs)
The present invention relates to the field of therapeutic use of leptin, leptin analogs, and leptin derivatives for the treatment of human adipose tissue atrophy.

All references cited herein are incorporated herein by reference. All cited references are listed at the end of the detailed description.

Adipose tissue atrophy (also known as lipodystrophy) syndrome is a diverse group of syndromes characterized by a deficiency of fat or adipose tissue. Metabolic disorders may also be associated with this condition. These metabolic disorders usually include hypertriglyceridemia and severe insulin resistance associated with diabetes (Reitmann et al., 2000). Adipose tissue atrophy in humans can be hereditary or acquired. There are two or more genotypes of adipose tissue atrophy. For example, mutations in the gene encoding Lamine A / C (LMNA) have been suggested to be associated with Danigan-type familial partial lipodystrophy (FPLD) (Cao et al., 2000). Individuals with Danigan FPD are born with a normal fat distribution, but during puberty, except for visceral and head and neck adipose tissue, develop progressive deficiency of subcutaneous limbs and trunk fat. Different chromosomal regions (9p34) are also associated with disease genes for congenital systemic lipodystrophy (Garg et al., 1999). Congenital systemic lipodystrophy is a recessive genetic disorder characterized by a near-complete deficiency of adipose tissue from birth, insulin resistance, hypertriglyceridemia and epidermolysis bullosa.

Some types of human adipose tissue atrophy are acquired. For example, many patients infected with human immunodeficiency virus (HIV) and treated with highly active antiretroviral therapy (HAART) are similar to those observed with increased visceral fat and Cushing syndrome. It produces partial lipodystrophy characterized by a lack of subcutaneous fat in the face, limbs and body, accompanied by a "buffalo hump". These patients may also develop metabolic disorders such as insulin resistance and hypertriglyceridemia. Acquired forms of adipose tissue atrophy may also be associated with juvenile dermatomyitis and other autoimmune diseases.

Studies in animal models have revealed that these metabolic disorders may be associated with fat deficiency (Gavrilova et al., 2000). However, insulin resistance and hypertriglyceridemia, which are characteristic of adipose tissue atrophy, were highly refractory to treatment despite various attempts (Garg, 2000). One of these techniques is treatment with thiazolidinedione, a PPARγ (peroxisome proliferator-activated receptor γ) agonist. While thiazolidinedione is attractive because it promotes both adipocyte differentiation and insulin sensitivity, patients receiving thiazolidinedione receive high doses of insulin, oral hypoglycemic substances (eg, metformin and thiazolidinedione), and lipid-lowering drugs. It is usually managed with combination therapies containing (eg, fibrates and statins). Despite these treatments, patients with systemic adipose tissue atrophy have severe hypertriglyceridemia (causing relapse of acute pancreatitis), severe hyperglycemia (diabetic retinopathy and nephropathy). (Introducing danger), and the symptoms of non-alcoholic steatohepatitis (which can result in cirrhosis) continue (Arioglu et al., 2000). In fact, one of the thiazolidinediones, troglitazone, has been removed from the US market due to rare but severe hepatocytotoxicity, and only two thiazolidinediones (rosiglitazone and pioglitazone) are commercially available (Reitmann, et al.). Therefore, there is a need for alternative treatments for adipose tissue atrophy.

Various genetic laboratory animal models for adipose tissue atrophy have been developed and tested. However, these models provide inconsistent results regarding susceptibility to treatment with leptin. For example, in one transgenic mouse model that expresses a cleaved nucleic acid variant of SREBP-1c and is characterized by congenital systemic lipodystrophy with insulin resistance and markedly low adipose tissue, continuous systemic leptin. Administration overcame the resistance of mice to insulin (Shimomura et al., 1999). On the other hand, in different transgenic mice expressing the A-ZIP / F-1 gene and characterized by adipose tissue deficiency, severe resistance to insulin, diabetes and highly reduced serum leptin levels, comparable doses. It did not respond to leptin and was less effective at higher doses (Gavrilova et al., 2000). The efficacy of leptin also diminished with age of the animal (supra). In addition, insulin resistance was overcome with leptin in SREBP-1c transgenic mice, but no recovery from adipose tissue atrophy was observed (Shimomura et al.).

WO00 / 20872 WO 96/05309 WO 96/40912 WO 97/06816 WO 97/18833 WO 97/38014 WO 98/08512 WO 98/28427 US 5,521,283 US 5,525,705 US 5,532,336 US 5,552,522 US 5,552,523 US 5,552,524 US 5,554,727 US 5,559,208 US 5,563,243 US 5,563,244 US 5,563,245 US 5,567,678 US 5,567,803 US 5,569,743 US 5,569,744 US 5,574,133 US 5,580,954 US 5,594,101 US 5,594,104 US 5,605,886 US 5,614,379 US 5,691,309 US 5,719,266 WO96 / 23513 WO96 / 23514 WO96 / 23515 WO96 / 23516 WO96 / 23517 WO96 / 23518 WO96 / 23519 WO96 / 34111 WO 96 37517 WO96 / 27385 WP 97/00886 EP 725578 EP 725079 EP 744408 EP 745610 EP 835879 WO 96/22308 WO96 / 31526 PCTWO96 / 34885 WO 97/46585 WO 96/35787 WO97 / 16550 WO 97/20933 EP 736599 EP 741187

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Leptin in human therapy is currently used primarily to reduce obesity and its associated metabolic dysfunction (Heymsfield et al. 1999). Leptin-deficient patients due to mutations in the leptin gene are morbidly fat from early childhood and have many hormonal abnormalities, including insulin resistance and hypogonadotropin dysfunction (Montague et al., 1997). .. One year of physiological replacement of recombinant leptin in one of these patients resulted in significant weight loss and amelioration of hormonal abnormalities (Farooqi et al., 1999; PCT International Application No .: WO 00/20872). These previous studies have not examined the use of leptin associated with human adipose tissue atrophy.

The present invention provides a method for determining the use of leptin in the treatment of humans suffering from adipose tissue atrophy and associated metabolic disorders, and the predisposition to leptin treatment. In one embodiment, human leptin is used in hormone replacement therapy in patients with adipose tissue atrophy with reduced leptin serum levels. Preferably, recombinant human leptin or leptin analogs or derivatives are used. Leptin proteins can be administered subcutaneously or systemically, or via any other route, including methods in gene therapy.

To determine the predisposition of patients with adipose tissue atrophy to treatment with leptin, the serum concentration of leptin can be measured. A patient having a serum leptin concentration of preferably 4 ng / ml or less, more preferably 2 ng / ml or less, and optimally 0.5 ng / ml or less is in a state of undergoing leptin treatment. Treatment with leptin is also preferably given to female patients with serum leptin concentrations below 4 ng / ml and to male patients with serum leptin concentrations below 3 ng / ml. More preferably, leptin is given to male patients with serum leptin concentrations below 2 ng / ml.

FIG. 1A shows the clinical course of patient NIH-1 with 4 months of leptin treatment. Pre-leptin treatment sources (starting on day 0) are presented to prove the rigor of metabolic discoveries. Significant outcomes of improvement in therapeutic and metabolic parameters are shown. FIG. 1B shows a T1-weighted nuclear magnetic resonance image of patient NIH-1 at L4 levels with reference and 4 months of leptin treatment. Note the decrease in liver size and the inevitable changes in the position of the kidneys and midline structure.

FIG. 2 shows that leptin reduces HbAic in diabetic patients (n = 8). The data showed error bars showing mean change and 95% confidence intervals. References and 4-month ± SEM values (mean standard error) are also shown. * P <0.001.

FIG. 3 shows that leptin improves the glucose curve during both insulin resistance and oral glucose tolerance (n = 9). Panel A: 0.2 U.S. before leptin treatment (black circles and lines) and 4 months later (white circles and dotted lines). Plasma glucose in response to kgIV insulin. Error bars indicate SEM. * P <0.02. Panel B: Plasma glucose in response to 75 grams of oral glucose before (black circles and lines) and 4 months after leptin treatment (white circles and dotted lines). Error bars indicate SEM. * P <0.01.

FIG. 3 shows that leptin improves the glucose curve during both insulin resistance and oral glucose tolerance (n = 9). Panel A: 0.2 U.S. before leptin treatment (black circles and lines) and 4 months later (white circles and dotted lines). Plasma glucose in response to kgIV insulin. Error bars indicate SEM. * P <0.02. Panel B: Plasma glucose in response to 75 grams of oral glucose before (black circles and lines) and 4 months after leptin treatment (white circles and dotted lines). Error bars indicate SEM. * P <0.01.

FIG. 4 shows that leptin reduces triglycerides. The data showed error bars showing the mean change from the reference and the 95% confidence interval. The criteria for the observed range and the 4-month mean are also shown. Note that the data are asymmetric and not normally distributed. * P <0.001.

The adipocyte hormone leptin plays a central role in energy homeostasis. Leptin was first discovered in obese mice as an unknown serum factor that reduced food intake and body weight upon replacement (Zhang et al., 1994; Pelleymounter et al., 1995). For these first observations, many of the initial treatment attempts with this hormone are obesity treatments. Many obese individuals have high serum leptin levels and may have a state of leptin resistance (Mantzoros et al., 2000). So far, the effects of recombinant human leptin have been limited to cause weight loss in obese individuals, except in the case of congenital leptin deficiency (Heymsfield et al., 1999; Farooqi et al., 1999).

The present invention includes adipose tissue atrophy and hyperglyceridemia, abnormal lipidemia, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, atherosclerosis, vascular restenosis and insulin resistance in humans It provides the possibility of using leptin for the treatment of metabolic disorders associated with the atrophy. Studies in HIV patients show that a decrease in leptin serum levels is closely associated with the development of acquired adipose tissue atrophy. In addition, leptin replacement in patients with adipose tissue atrophy dramatically improves glucose and triglyceride metabolism, even after all other possible treatments have been discontinued. In all of these leptin replacement therapies, the reference serum concentration of leptin was less than 4 ng / ml.

In one severe case of acquired adipose tissue atrophy, the patient (having a serum leptin concentration of less than 0.5 ng / ml) had severe hypertriglyceridemia, diabetes, painful eruptive cutaneous xanthoma and giant liver. He had a tumor. Leptin treatment for more than 4 months dramatically improved hypertriglyceridemia and hyperglycemia in patients and discontinued plasmapheresis and other diabetic medications. The improvement was also accompanied by the disappearance of cutaneous xanthoma and a 40% reduction in the patient's liver volume. Therefore, these data indicate that leptin replacement therapy can be effectively used for the treatment of acquired or congenital adipose tissue atrophy and associated metabolic disorders in humans.

Furthermore, based on these data, it can be estimated that patients with leptin serum concentrations below 4 ng / ml may be a suitable group of patients for replacement therapy with leptin. Leptin levels can be measured using body fluids, preferably blood or any portion thereof. In this specification, individual sera were used. Other body fluids such as whole blood, cerebral spinal cord secretions, plasma and preferably urine may also contain measurable leptin. A measurement of 4 ng of leptin per 1 ml of serum can correlate with the corresponding level in other body fluids. For example, when whole blood is used, the leptin concentration is diluted to take into account the diluting effect of the unfractionated blood used.

Those skilled in the art can confirm the effective dose by administering leptin, a leptin analog or a leptin derivative and observing the desired therapeutic effect. The goal of replacement therapy is to achieve near the physiological concentration of leptin in plasma. Physiological replacement doses of leptin were approximately 0.02 mg / kg bw / day for men of all ages, approximately 0.03 mg / kg bw / day for women under the age of 18, and approximately 0. It is estimated to be 04 mg / kg body weight / day. To achieve near physiological levels of leptin, for example, 50% of the estimated replacement dose in the first month of treatment, 100% of the replacement dose in the second month of treatment, in the third month of treatment. The patient may be treated with 200% of the replacement dose. During leptin replacement therapy, any biochemical label can be measured to monitor the therapeutic effect of leptin treatment. Glycosylated hemoglobin (HbAic) and triglyceride (fasting) levels are suitable labels for monitoring the effectiveness of leptin treatment and measuring therapeutic efficacy.

Alternatively, serum leptin levels can be measured using commercially available immunoassays, as further shown in the Examples below. In general, diagnostic analysis for measuring the amount of leptin in blood (or plasma or serum) can first be used to determine the intrinsic level of protein. Such diagnostic means may be in the form of antibody analysis, such as antibody sandwich analysis. The amount of endogenous leptin is first measured to determine the criteria. Therapeutic doses are determined as quantification of internal and external leptin proteins (ie, either self-produced or administered, leptin, leptin analogs or leptin derivatives observed in the body). Observation of the patient's leptin levels continued during the course of treatment.

The present invention also provides a method of using a pharmaceutical composition of leptin, a leptin analog or a leptin derivative. Such pharmaceutical compositions can be infused or administered orally, pulmonary, nasally, percutaneously or in other forms of administration. Suitable methods of administration of leptin protein include subcutaneously systematic or gene therapy.

In general, the pharmaceutical compositions of the present invention provide an effective amount of leptin, leptin analog or leptin derivative with a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, excipient and / or carrier. Including. Such compositions include various buffer content diluents (eg, Tris-HCl, acetates, phosphates), pH and ionic strength diluents; purifiers and solubilizers (eg, Tween 80). , Polysolvate 80), additives such as antioxidants (eg, ascorbic acid, sodium metabisulfite), preservatives (eg, Thimersol, benzyl alcohol) and fillers (eg, lactose, mannitol); polylactic acid, Incorporation of substances into particle formulations or liposomes of polymer compositions such as polyglycolic acid. Hylauronic acid, which can have a sustained duration-promoting effect in circulating blood, can also be used. Such compositions may affect the physical condition, stability, rate of in vivo release, and rate of in vivo removal of the protein and derivatives. See, for example, pages 1435-1712 of Remington's Pharmaceutical Sciences, 18thEd. (1990, Mack Publishing Co., Easton, PA 18042), attached as a reference herein. The composition can be prepared in a liquid form or in a dry powder such as a lyophilized form. Implantable sustained release forms can also be considered as transdermal forms.

Surfactants have been added as wetting agents to assist in the decomposition of the therapeutic material into the aqueous environment. Surfactants may include anion purifiers such as sodium lauryl, dioctyl sulfosuccinate and sodium dioctyl sulfonate. A cation purifier could be used and could contain benzalkonium chloride or benzthionium chloride. A list of potential nonionic purifiers that may be included in the form as surfactants is lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrohardened castor oil 10, 50 and 60, glycerol monostearate, Polysorbates 40, 60, 65 and 80, fatty acid ester sucrose, methyl cellulose and carboxymethyl cellulose. These surfactants can be present in either the form of proteins, or derivatives alone or in mixtures of different ratios.

Additives that potentially enhance the intake of leptin, leptin analogs or leptin derivative proteins include, for example, fatty acids, oleic acid, linoleic acid and linolenic acid.

A controlled release format may be desirable. Leptin, leptin analogs or leptin derivative proteins could be incorporated into inactive matrices such as gum, allowing release by either diffusion or elution mechanism. Slowly degenerate matrices can also be incorporated into forms such as alginate, polysaccharides. Another form of controlled release of the treatment is, for example, a semi-leptin, leptin analog or leptin derivative protein that allows osmotic effects to allow water to enter and the protein to be extruded through a single small hole. It is a method based on the Oros therapeutic system (Alza), which is surrounded by a permeable thin film. Some enteric coatings also have a delayed release effect.

In addition, an improved kit for determining the predisposition to human patients with adipose tissue atrophy in response to treatment with leptin, leptin analogs or leptin derivatives is examined by the present invention. In one aspect, the modified kit can provide a means for determining whether the leptin level of the patient prior to the leptin treatment is about 4 ng / ml or less. In a related aspect, the modified kit can take into account the patient's gender when determining the patient's leptin levels prior to the leptin treatment. Thus, the kit may provide a means for determining whether a patient's leptin level prior to leptin treatment is about 2 ng / ml or less if the patient is male and about 4 ng / ml or less if the patient is female. it can. Preferably, the kit includes an instruction manual. The kit may further include reagents, tubes, packaging and / or other reaction compositions.

The following description is provided as an example only and does not limit the scope of the claims and should not be construed as such. Based on the description, those skilled in the art may make modifications and modifications to suitable embodiments not beyond the scope of the present invention.

Example 1
The following examples show that the progression of HIV-related adipose tissue atrophy syndrome (HIV-LS) can be affected by decreased leptin in the serum that contributes to the accumulation, deficiency or redistribute of body fat.

Specifically, this study was conducted to determine whether the adipose tissue atrophy phenotype of HIV-LS is associated with changes in serum leptin following the initiation of highly active antiretroviral therapy (HAART). .. The study included 146 (146) HIV-positive men whose serum leptin levels were compared before and after HAART. Physical examination evaluated and stratified men into two major phenotypes: adipose tissue atrophy alone, and adipose tissue atrophy with central fat gain (“mixed” HIV-LS).

Forty-two of the 146 men (42/146) were found to have moderate or severe adipose tissue atrophy or hypertrophy in one or more body areas after HAART. Twenty-seven of 146 (27/146) had adipose tissue atrophy alone and 15 of 146 (15/146) changed to "mixed" after HAART. Thirty-nine of 146 patients (39/146) had no changes in physical constitution and these patients were used as controls. In general, men with HIV-LS were older and had longer-term use of protiase inhibitors. They had a lower reference CD4 count and lost an average of 4 kg from the baseline.

Prior to HAART, both the adipose tissue atrophy and the "mixed" groups had a median reference leptin level of 3.6 ng / ml and a control median leptin level of 4.1 ng / ml. In individuals who developed only adipose tissue atrophy after HAART, serum leptin levels were significantly reduced from 3.6 to 2.8 ng / ml (Wilcoxon p = 0.06). On the other hand, of the "mixed" HIV-LS group (4.0 ng / ml) [p = NS] and 39 HIV-positive controls (3.7 ng / ml) [p = NS] who did not express HIV-LS. In both, serum leptin levels remained stable.

These data suggest that a decrease in leptin levels following high-activity antiretroviral therapy in HIV-positive patients may contribute to the progression of adipose tissue atrophy syndrome.

Example 2
To determine the efficacy of using leptin to treat adipose tissue atrophy in humans, leptin replacement therapy was also given to nine female patients diagnosed with various forms of adipose tissue atrophy. Patients in this study were investigated by a number of physicians in the United States and Europe. To qualify, patients must have low levels associated with adipose tissue atrophy (defined as serum leptin concentrations <3.0 ng / ml in men and <4.0 ng / ml in women), and It was requested to be accompanied by at least one abnormal metabolism as follows. : It is (1) the presence of diabetes by the American Association of Diabetes (see Peters et al. 1998); (2) fasting serum triglyceride levels above 200 mg / dL; and / or (3) fasting serum insulin levels. Is higher than 300 μU / ml. Diagnosis of adipose tissue atrophy was made based on clinical viewpoints well known to those skilled in the art.

Table 1 outlines the baseline clinical characteristics of the patients treated in this study.

The nine patients enrolled in this study were all female. Although this study is available for both genders, women tend to be recognized earlier and more often. Five of the nine patients had congenital generalized adipose tissue atrophy or Seip-Beradinelli syndrome. This analysis was defined by evidence of generalized fat deficiency from birth associated with other clinical criteria (Online Mendelian Inheritance in Man, OMIM # 269700; Garg et al., 1992). It was found that three patients had acquired systemic lipodystrophy in the history of apparent fat deficiency in childhood. One of these patients (UTSW-2) developed a generalized lipodystrophy with juvenile dermatomyitis. Another patient (NIH-7) had Danigan familial systemic lipodystrophy (OMIM # 151660; Garg, 1999; and Cao et al., 2000).

Study Design This study was designed as an expected open-label study at the Diabetes Division of the National Institute for Diabetes, Digestive and Kidney Diseases (NIDDK), and at the UT Southwestern Medical Center in Dallas. Amgen Inc. (Thousand Oaks, CA) provided recombinant thionyl human leptin (recombinant leptin) as an attempt. The response of each patient was compared to their reference condition. Due to the rarity of adipose tissue atrophy syndrome and the variability of clinical characteristics, it was not feasible to include a randomized, placebo-treated control group. NIDDK and the Institutional Ethics Committee of the University of Texas Southwestern Medical Center approved the study. Written consent was obtained from the patient or legal guardian.

Patients were considered as inpatients before and one, two, and four months after leptin treatment again at the National Institutes of Health Clinical Center and the University of Texas Southwestern Medical Center General Clinical Research Center. All patients were on stable doses of the concomitant drug for at least 6 weeks prior to starting leptin. During this study period, hypoglycemic drugs were gradually reduced or discontinued at any time.

The goal of this study was to achieve near-physiological levels of leptin in plasma. Physiological replacement doses are estimated to be 0.02 mg / kg / day for men of all ages, 0.03 mg / kg / day for women under 18 years and 0.04 mg / kg / day for adult women. It was. Recombinant leptin was administered subcutaneously every 12 hours. It is important to note that the replacement dose is about one tenth of the most commonly used dose in obesity studies. Patients were treated with 50% of the replacement dose during the first month, 100% replacement dose the following month, and 200% replacement dose during the next two months. The primary endpoints that determine the effect of recombinant leptin were determined as hemoglobin Aic and fasting serum triglyceride levels.

Biochemical analysis Serum glucose and triglyceride levels were determined by standard methods using automated Hitachi equipment (Boehringer Mannheim, Indianapolis, IN) and Beckman equipment (Beckman, CA). Hemoglobin Aic was determined by ion exchange high performance liquid chromatography (Bio-Rad Laboratories, Heracles, CA). Serum free fatty acid (FFA) levels were determined with a commercial kit (Wako, Richmond, VA). Serum insulin levels were determined by immunoassay using reagents provided by the Abbott Imx instrument (Abbott Park, IL) and a commercial kit (Linco Research, Inc., St. Charles, MO). Serum leptin levels were determined by immunoassay using a commercial kit (Linco Research, Inc. St. Charles, MO).

Procedure Resting energy consumption was measured using the Deltatrac facility (Sensormedics, Yorba Linda, CA). The study was performed in resting patients who arose between 6 and 8 am after an overnight fast of at least 8 hours. The oral glucose tolerance test was performed after an overnight fast with 75 g of glucose. Serum glucose was measured at -10, 0, 30, 60, 90, 120 and 180 minutes of glucose loading.

High-dose insulin resistance studies were performed with 0.2 IU / kg adjusted insulin to assess insulin sensitivity. Insulin was administered intravenously after an overnight fast. Samples for glucose were collected at -10, 0, 5, 10, 15, 20 and 30 minutes of insulin administration. The K constant (the rate of glucose deficiency as a reflection of systemic insulin sensitivity) was calculated using first-series kinetics as a constant rate of blood glucose drop after intravenous insulin (Harrision et al., 1976).

Body fat was determined using a dual-energy X-ray absorption spectrometer (DEXA, Hologic QDR 4500) (Hologic, Inc., Bedford, MA) (Lambrinoudaki et al., 1998). Tl-axis weighted MR scans of the liver were obtained on a 1.5 Tesla scanner (General Electric Medical Systems, Milwaukee) (Abate et al., 1994). Liver volume was calculated on a sun workstation using the MEDx image analysis software package (Sensor Systems, Inc., Sterling, VA). Fluorescent views of the outer phase of the liver were made on individual contact slices by determining the tip of the algorithm below. Liver volume was then calculated based on pixel area and slice thickness. Subjects participating in the NIH department were asked to report the criteria for the last 3 days and food intake after 4 months to calculate the estimated daily food intake (Feskanich et al., 1993). ..

Statistical analysis Measured values are shown as mean ± SEM. Repeated means ANOVA was used to compare study variables over different study periods. Asymmetric data such as triglyceride concentration and measured K constants were logarithmically converted. The paired t-test was used to compare reference data with as many time points as applicable. Plasma glucose levels during the oral glucose tolerance test period were compared using a two-factor analysis of variance for the study period and time in the test period modeled as repeated factors. The 95 percent confidence interval for the difference between the means came from analysis of variance and the difference between the means (Hanh et al., 1991). The conversion was considered statistically significant at p <0.05. Adjustments for simultaneous comparisons were not made for statistical analysis of special priority hypotheses.

Results Criteria Patient characteristics In this study, 8 out of 9 patients had diabetes and all had hyperlipidemia (Table 1). All diabetic patients received drug therapy prior to this study (Tables 1 and 2), and 4 patients received drug therapy for fat management (Table 1). The average HbAic of diabetic patients was 9.1 ± 0.5% (usually: <5.6%). Average triglyceride levels were elevated to 1405 mg / dL (range: 322-7420 mg / dL; normal range: 35-155 mg / dL) [16 mmol / L, range: 3.6-8.7 mmol / L]. Free fatty acid (FFA) levels were increased approximately three-fold of the normal upper limit (1540 ± 407 μmol / L; normal: 350-550 μmol / L). Six of the seven NIH patients had fatty liver on ultrasound and hypertrophied liver on physical trials. Three of the patients underwent liver biopsy and two of the three were diagnosed with non-alcoholic steatohepatitis based on histopathological criteria. (Manton et al., 2000; Berasain et al., 2000; Luyckx, et al., 2000).

The average serum leptin concentration was 1.3 ± 0.3 ng / mL according to the standard (Table 1), 2.3 ± 0.5 ng / mL at the end of the first month of treatment, and 5. at the end of the second month. Increased with treatment up to 5 ± 1.2 ng / mL and 11.1 ± 2.5 ng / mL at the end of April. Therefore, the dose of recombinant leptin used in this study gives near-normal serum leptin levels in these patients.

Leptin effect of first patient: case study (Fig. 1)
The first patient treated in this study (NIH-1) was the most severely affected, and her course of leptin replacement in this population even after discontinuation of all other possible treatments. It is suggestive to show a dramatic effect. This patient was born healthy but experienced a fat deficiency between the ages of 10 and 12. She developed severe hypertriglyceridemia at age 13 and diabetes at age 14. She visited the NIH Clinical Center at the age of 15 with diabetes with consistently higher triglyceride levels than 10000 mg / dL (higher than 113 nmol / L) and 9.5% HbAic. She had scattered painful eruptive cutaneous xanthomas throughout the giant hepatomegaly that extended to the edges of the body and pelvis. Weekly plasmapheresis and Orlistat were added to reduce hypertriglyceridemia (Fig. 1A) (Bolan et al). Other prominent clinical features included a great appetite (she reported eating above 3200 kcal / day) and a very high resting metabolic rate of 2010 kcal / day, which is 180% of the predicted value. Recombinant leptin for more than 4 months caused significant progressive improvements in hypertriglyceridemia and hyperglycemia due to plasmapheresis and discontinuation of diabetic drugs (Fig. 1A). Improvements in metabolic parameters were accompanied by the disappearance of cutaneous xanthoma. In addition, her liver volume decreased by 40% (from the reference 4213 mL shown in FIG. 1B to 2644 mL after 4 months).

Leptin improved metabolic control in all diabetic adipose tissue atrophy patients Prior to the initiation of leptin treatment, eight diabetic adipose tissue atrophy patients had weak metabolic control. After 4 months of leptin replacement treatment, HbAic decreased by an average of 1.9 percentage points (95% CI, 1.1-2.7%, p = 0.0012) (Fig. 2). The individual reactions of the patients are shown in Table 3. Criteria for reduction or discontinuation It is noteworthy that glycemic control improved despite antidiabetes treatment (Table 2).

Plasma glucose levels during the insulin resistance test period showed a significant improvement compared to the baseline at the end of 4 months (Fig. 3A). The K value (rate of glucose loss) increased from 0.0071 ± 0.0012 to 0.0169 ± 0.0039, indicating improved systemic insulin sensitivity (p = 0.035). In addition, oral glucose tolerance was also significantly improved compared to the standard (Fig. 3B).

At the end of 4 months of recombinant leptin treatment, fasting triglyceride levels dropped to 60% (CI, 43-77%, p <0.001, FIG. 4). During this same period, fasting free fatty acids dropped from 1540 ± 407 μmol / L to 790 ± 164 μmol / L (p = 0.045). The individual reactions are shown in Table 3.

Changes in liver volume and liver function tests The standard mean liver volume was 3097 ± 391 mL (approximately 4-fold increase in age- and gender-matched normal body weight individuals). Leptin reduced liver volume by an average of 28% (CI, 20-36%) from baseline. The average decrease in liver volume was 987 mL (CI, 546 to 1428 mL, p = 0.0024). Improvements in liver size were associated with improvements in liver function testing. The reference alanine transaminator concentration decreased from 66 ± 16 U / L to 24 ± 4 U / L at the end of 4 months (p = 0.023). Similarly, serum aspartate aminotransferase concentrations were 53 ± 12 U / L at the reference and 21 ± 2 U / L at the end of 4 months (p = 0.03).

Changes in energy balance Self-reported daily calorie intake was significantly reduced from the standard of 2680 ± 250 kcal / day to 1600 ± 150 kcal / day (p = 0.005, n = 7). There was a parallel decrease from an accurate resting metabolic rate of 1920 ± 150 kcal / day to 1580 ± 80 kcal / day (p = 0.003, n = 9).

All but one subject (NIH-3) had lost weight at the end of April or the end of the month. The average weight loss was 3.6 ± 0.9 kg in the range between -1.7 and 7.3 kg. An important part of weight loss (50-65%) can be due to loss of liver weight.

Tolerance and side effects No skin reaction at the injection site was reported or observed. There was no tendency for side effects on the given biochemical or blood parameters. Patient NIH-1 had nausea and vomiting attacks after the first dose. Patient NIH-6 had exacerbation of hypertension after a second dose associated with lavage.

Patient NIH-7 was hospitalized for streptococcal infection during the third month of treatment. None of these events recurred with continued treatment.

Discussion In this study, leptin substitution provided clear and dramatic metabolic benefits in a group of patients with lipodystrophy and leptin deficiency. During this study, replacement with recombinant leptin improved HbAic by 1.9 percentage points, which reduced the associated risk of developing retinopathy in ~ 22% of the diabetic population (PDS, UK, 1998). It is estimated to be. In addition, triglyceride levels are reduced to 60%, which is estimated to reduce the relative risk for cardiovascular events throughout the population to 35-65% (Kreisberg, 1998; Garg, 2000). ).

These results provide new insights into the mechanism of action of leptin. Leptin signals appear to regulate systemic insulin sensitivity and triglyceride levels, in addition to their known role in controlling energy homeostasis. This study is the first evidence that leptin functions in vivo as an insulin sensitizer and insulin reserve in humans.

Although we did not use a randomized study design, the importance of the evidence suggests that improved metabolic regulation was caused by leptin rather than improved compliance of participants in the study. First, the magnitude and reproducibility of HbAic improvement is most consistent with drug results rather than placebo effects. Despite the patient asymmetry included in this study, we observed a constant improvement in metabolic control in all diabetic patients. Patient NIH-2 has evidence of non-compliance, which is explained by the deterioration of her HbAic during the second and April, corrected by long-term treatment (Table 3). Improved drug discontinuation in this patient is strong evidence that improved HbAic levels are the effect of leptin administration.

Effect of Leptin on Food Intake Restriction of caloric intake in adipose tissue atrophic diabetes has been recognized to improve glucose and dyslipidemia (Trygstad et al., 1977). However, patients have difficulty following dietary restrictions due to their appetite. Leptin clearly reduced food intake in these patients. A limited study was performed on patient NIH-1 to determine the contribution of reduced food intake on metabolic parameters. At the hospital, she discontinued leptin for 9 days and fixed her caloric intake to pre-discontinuation levels. Despite being on a stable diet, her fasting insulin, triglyceride and glucose levels increased within 48 hours. These observations indicate that leptin has an effect on insulin sensitivity and triglyceride metabolism, independent of its effect on food intake. Similar data have been reported using pair-feeding experiments in adipose tissue atrophic mice with and without leptin (Shimomura et al., 1999; Ebihara et al., 2001). ).

Correlation with Mouse Models Various mouse models of adipose tissue atrophy suggested that the absence of adipose tissue was responsible for insulin resistance in this syndrome (Burant et al., 1997; Moitra et al., 1998; Shimomura et al., 2000). The proof that adipose tissue transplantation dramatically improves insulin resistance and metabolic regulation in adipose tissue atrophic mice strongly supports this hypothesis (Gavrilova et al., 2000). However, it remains unclear why adipose tissue is needed to maintain systemic insulin sensitivity. The observations and results discussed above, along with Shimomura et al., Supra, suggest that most of the regulatory effects of adipose tissue on systemic insulin sensitivity are mediated by leptin.

The possible mechanism by which leptin regulates both insulin sensitivity and lipid metabolism may be based on SREBP1c, a transcription factor that stimulates lipid production. In the liver, SREBP1c is upregulated by the hyperinsulinemia found in adipose tissue atrophy. Leptin deficiency and hyperinsulinemia cause down-regulation of the insulin receptor substrate IRS-2, impair insulin action and increase hepatic glucose production. Increased lipogenesis and hepatic glucose production cause a vicious cycle. Increased tissue lipid levels are associated with decreased systemic insulin sensitivity and additional hepatic glucose production. Leptin substitution has been shown to correct for this vicious cycle. Although the rate of triglyceride synthesis has not been studied in humans with adipose tissue atrophy, indirect calorimetric studies provide some evidence that lipogenesis can actually be unregulated (Arioglu et al.,, 2000). Other observations showed reduced resting energy expenditure in patients treated in this study. This may be due to reduced food intake, which results in reduced diet-induced heat production.

Leptin: Antilipidemia Hormone
Leptin administration in Zucker rats has been reported to lead to correction of steatosis in various organs that function as lipid accumulation sites; it is at sites such as the islet cells of the liver or heart cells (Unger, 1995; Unger et al., 1999). Lipid accumulation outside of adipocytes may spill over into phenomena caused by adipocytes that have reached their maximum volume of triglyceride storage. In lipodystrophy, these organs are the only sites that can store lipids. Leptin treatment in mice with lipodystrophy causes a dramatic reduction in hepatic triglyceride storage. In parallel, leptin treatment in humans suffering from lipodystrophy causes a significant and very large reduction in liver volume.

Timing for Leptin Replacement The concept that adipose tissue is an endocrine organ was strongly supported by the discovery of leptin. Leptin has both direct and / or indirect effects in major metabolic organs, including the brain, liver, muscle, fat and pancreas. Leptin is certainly not the only circulating adipocyte signal. For example, other adipocyte hormones include adipocyte-specific composition-related protein (ACRP) 30 / adiponectin / AdipoQ, which appears to be important in inducing fatty acid formation in muscle and liver (Yamauchi et al., 2001; Fruebis et al., 2001; Berg et al., 2001). If a fat cell deficiency results in a deficiency in all known and undiscovered fat-inducing signals, it therefore contributes to many of the abnormalities seen in syndromes characterized by fat deficiency. This study is the first human study to judge from the metabolic effect of replacing fat-inducing hormones in the state of fat deficiency. Leptin deficiency has been shown to contribute primarily (but not only) to the metabolic disorders associated with adipose tissue atrophy. Therefore, this study highlights an important reason to consider leptin replacement therapy in humans; that is, severe lipodystrophy.

Example III
The amino acid sequence for mature recombinant methionyl human leptin is shown herein as SEQ ID NO: 1, where the first amino acid of the mature protein is valine (position 1) and the methionyl residue is position -1 (position 1). It is located in rHu-Leptin 1-146, defined herein as SEQ ID NO: 1).

Alternatively, a naturally occurring variant of human leptin with a 145 amino acid with a glutamine deficiency at position 28 compared to rHu-leptin 1-146 shown below can be used (as defined herein, rHu-leptin 1). -145, defined as SEQ ID NO: 2, where the blank "*" indicates the absence of amino acids).

Other examples of leptin proteins, analogs, derivatives, preparations, dosage forms, pharmaceutical compositions, dosages and routes of administration were previously described in the following PCT application and are described herein in full below. As shown, it is shown herein for reference. PCT International Application Nos. WO 96/05309; WO 96/40912; WO 97/06816; WO 00/20872; WO 97/18833; WO 97/38014; WO 98/08512 and WO 98/28427.

Leptin proteins, analogs and related molecules have also been reported in the following publications; however, there is no description of the action of any of the reported compositions.
US Patent Number US 5,521,283; US 5,525,705; US 5,532,336; US 5,552,522; US 5,552,523; US 5,552,524; US 5,554,727; US 5,559,208; US 5,563,243; US 5,563,244; US 5,563,245; US 5,567,678; US 5,567,803; US 5,569,743; US 5,569,744; 5,574,133; US 5,580,954; US 5,594,101; US 5,594,104; US 5,605,886; US 5,614,379; US 5,691,309; US 5,719,266 (Eli Lilly and Company);
PCT WO96 / 23513; WO96 / 23514; WO96 / 23515; WO96 / 23516; WO96 / 23517; WO96 / 23518; WO96 / 23319; WO96 / 34111; WO 96 37517; WO96 / 27385; WP 97/00886; EP 725078; EP 725079; EP 744408; EP 745610; EP 835879 (Eli Lilly and Company);
PCT WO96 / 22308 (Zymogenetics);
PCT WO96 / 31526 (Amylin Pharmaceuticals, Inc.)
PCTWO96 / 34885; WO 97/46585 (SmithKline Beecham, PLC);
PCT WO 96/35787 (Chiron Corporation);
PCT WO97 / 16550 (Bristol-Myers Squibb);
PCT WO 97/20933 (Schering Corporation)
EP 736599 (Takeda);
There is EP 741187 (F. Hoffman La Roche).

It can be used with the method within the scope of these references provided for useful leptin proteins or analogs, or related compositions or methods, such compositions and / or methods. Under the above conditions, these publications are presented herein for reference.

Example IV
Standard enzyme-linked immunosorbent assay (ELISA) can be used to determine serum leptin levels in patients with adipose tissue atrophy according to one embodiment of the invention. The ELISA method can use purified rat monoclonal anti-rmetHu-leptin antibody to capture leptin from serum. Horseradish peroxidase binding affinity purified rabbit anti-rmetHu-leptin polyclonal antibody can also be used to detect captured leptin. The detection limits of the analysis using these antibodies may be in the range of 0.5-0.8 ng / ml. Although some antibodies may have been used, the preferred antibodies react specifically with natural human leptin and are sensitive to detect leptin levels below 5 ng / ml serum.

Preferably, the timing for determining the patient's reference leptin level is 8-12 hours after a fast, such as in the morning. Reference leptin levels were not confused by elevated levels, eg, after a meal, or due to elevated leptin sleep cycles observed in most individuals (eg, elevated leptin levels at 3 am). Such reference levels may be used, for example, to observe elevated leptin levels at night, but those levels should be compared to similar levels in patients with similar conditions.

Based on the above data, the method of determining the predisposition of patients with adipose tissue atrophy to treatment with leptin is to determine the leptin level corresponding to the serum leptin concentration, and at a serum leptin concentration of about 4 ng / ml or less. By being, it can be done.

Claims (11)

A pharmaceutical composition containing a leptin protein for treating adipose tissue atrophy in humans or metabolic disorders associated with adipose tissue atrophy.
The leptin protein consists of the amino acid sequence of SEQ ID NO: 1 in which the methionyl residue is located at position -1.
All SANYO the composition is administered subcutaneously at a dose of leptin protein 0.01～0.08Mg / kg body weight / day,
Metabolic abnormalities associated with adipose tissue atrophy are selected from the group consisting of hyperglycemia, hypertriglyceridemia, fatty liver, steatohepatitis, hepatomegaly, and diabetes.
Pharmaceutical composition. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered to a patient having a leptin level of 4 ng / ml or less. The pharmaceutical composition according to claim 1 or 2, for administration to a patient having a leptin level of 2 ng / ml or less. The pharmaceutical composition according to any one of claims 1 to 3, wherein the adipose tissue atrophy is an acquired type. The pharmaceutical composition according to any one of claims 1 to 3, wherein the adipose tissue atrophy is a genotype. Claims 1 to 5 for treating metabolic abnormalities associated with adipose tissue atrophy selected from the group consisting of hyperglycemia, hypertriglyceridemia, fatty liver, steatohepatitis, hepatomegaly, and diabetes. The pharmaceutical composition according to any one. The pharmaceutical composition according to claim 6, wherein the metabolic disorder is hyperglycemia. The pharmaceutical composition according to claim 6, wherein the metabolic disorder is hypertriglyceridemia or diabetes. The pharmaceutical composition according to any one of claims 1 to 5, for treating adipose tissue atrophy. The pharmaceutical composition according to any one of claims 1 to 9, further comprising at least one compound selected from the group consisting of thiazolidinedione, fibrates, statins and metformin. The pharmaceutical composition according to any one of claims 1 to 10, which is administered at a dose of 0.02 to 0.08 mg / kg body weight / day of leptin protein.

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